Cytogenetic and dosimetric effects of (131)I in patients with differentiated thyroid carcinoma: comparison between stimulation with rhTSH and thyroid hormone withdrawal treatments.

A study directed to the cytogenetic and dosimetric aspects of radionuclides of medical interest is very valuable, both for an accurate evaluation of the dose received by the patients, and consequently of the genetic damage, and for the optimization of therapeutic strategies. Cytogenetic and dosimetric effects of (131)I in lymphocytes of thyroidectomized differentiated thyroid cancer (DTC) patients were evaluated through chromosome aberration (CA) technique: Euthyroid patients submitted to recombinant human thyroid-stimulating hormone (rhTSH) therapy (group A) were compared with hypothyroid patients left without levothyroxine treatment (group B). CA analysis was carried out prior to and 24 h, 1 week, 1 month and 1 year after radioiodine administration (4995-7030 MBq) in both groups. An activity-response curve of (131)I (0.074-0.740 MBq/mL) was elaborated, comparing dicentric chromosomes in vivo and in vitro in order to estimate the absorbed dose through Monte Carlo simulations. In general, radioiodine therapy induced a higher total CA rate in hypothyroid patients as compared to euthyroid patients. The frequencies of dicentrics obtained in DTC patients 24 h after treatment were equivalent to those induced in vitro (0.2903 ± 0.1005 MBq/mL in group A and 0.2391 ± 0.1019 MBq/mL in group B), corresponding to absorbed doses of 0.65 ± 0.23 Gy and 0.53 ± 0.23 Gy, respectively. The effect on lymphocytes of internal radiation induced by (131)I therapy is minimal when based on the frequencies of CA 1 year after the treatment, maintaining a higher quality of life for DTC patients receiving rhTSH-aided therapy.

Donepezil can improve ischemic muscle atrophy by activating angiomyogenic properties of satellite cells.

BACKGROUND: Saving more limbs of patients with peripheral arterial disease (PAD) from amputation by accelerating angiogenesis in affected limbs has been anticipated for years. We hypothesized that an anti-Alzheimer drug, donepezil (DPZ), can activate angiomyogenic properties of satellite cells, myogenic progenitors, and thus be an additional pharmacological therapy against PAD. METHODS AND RESULTS: In a murine hindlimb ischemia model, we investigated the angiogenic effects of a clinical dose of DPZ (0.2 mg·kg(-1)·day(-1)) and its combination with cilostazol, a platelet aggregation inhibitor and a conventional therapeutic drug against PAD. The combination therapy most effectively improved skin coldness and most effectively upregulated vascular endothelial growth factor (VEGF)-producing satellite cells in ischemic hindlimbs. Computed tomography revealed that DPZ remarkably attenuated ischemic muscle atrophy and induced super-restoration in affected hindlimbs. The in vitro study with human aortic endothelial cells showed that DPZ or its combination with cilostazol effectively upregulated the expression of pAkt, hypoxia inducible factor-1α, and VEGF protein. Likewise, in primary cultured satellite cells, DPZ, alone or in combination, upregulated the expression of VEGF, interleukin-1ß, and fibroblast growth factor 2 protein. CONCLUSIONS: The present results suggest that a clinical dosage of DPZ accelerates angiomyogenesis by directly acting on both endothelial and satellite cells. Therefore, DPZ is a potential additional choice for conventional drug therapy against PAD.

Effect of Brazilian propolis (AF-08) on genotoxicity, cytotoxicity and clonogenic death of Chinese hamster ovary (CHO-K1) cells irradiated with (60)Co gamma-radiation.

The present study was conducted in order to evaluate the effect of Brazilian propolis (AF-08; 5, 10, 15, 30, 50, 100, and 200µg/mL) in protecting CHO-K1 cells against genotoxic and cytotoxic damage and clonogenic death induced by (60)Co gamma-radiation (1.0, 2.0, 4.0, and 6.0Gy). For this purpose, three interlinked endpoints were analyzed: induction of DNA damage by use of the micronucleus (MN) test (genotoxic damage), cell viability by means of the MTS assay, and differential staining (cytotoxic damage) and clonogenic death via the colony-formation test (cytotoxic damage). The MN test revealed that propolis alone (5-100µg/mL) was not genotoxic up to 100µg/mL and that 30µg/mL of propolis reduced the radiation-induced DNA damage (∼56% reduction, p<0.05), exhibiting a radio-protective effect on irradiated CHO-K1 cells. On the other hand, analysis of cytotoxicity showed that a concentration of 50µg/mL presented a significant proliferative effect (p<0.001) when associated with radiation, decreasing the percentage of necrotic cells (p<0.01). No mediated cytotoxic effect was found, but the concentration of 200µg/mL was toxic when analyzed at 24 and 48h via the differential staining technique, but not at 72h after irradiation, analyzed with the MTS assay. Differential staining also showed that necrosis was the main death modality in irradiated cells and that apoptosis was induced only at the toxic concentration of propolis (200µg/mL). Concerning the clonogenic capacity, a concentration of 50µg/mL also exhibited a significant stimulating effect on cell proliferation (p<0.001), in agreement with the data from differential staining. Taken together, these data suggest that the use of propolis AF-08 for the prevention of the adverse effects of ionizing radiation is promising. Nevertheless, additional investigations are necessary for a better understanding of potential applications of propolis to improve human health.

Developmental toxicity, acute toxicity and mutagenicity testing in freshwater snails Biomphalaria glabrata (Mollusca: Gastropoda) exposed to chromium and water samples.

A protocol combining acute toxicity, developmental toxicity and mutagenicity analysis in freshwater snail Biomphalaria glabrata for application in ecotoxicological studies is described. For acute toxicity testing, LC50 and EC50 values were determined; dominant lethal mutations induction was the endpoint for mutagenicity analysis. Reference toxicant potassium dichromate (K2Cr2O7) was used to characterize B. glabrata sensitivity for toxicity and cyclophosphamide to mutagenicity testing purposes. Compared to other relevant freshwater species, B. glabrata showed high sensitivity: the lowest EC50 value was obtained with embryos at veliger stage (5.76mg/L). To assess the model applicability for environmental studies, influent and effluent water samples from a wastewater treatment plant were evaluated. Gastropod sensitivity was assessed in comparison to the standardized bioassay with Daphnia similis exposed to the same water samples. Sampling sites identified as toxic to daphnids were also detected by snails, showing a qualitatively similar sensitivity suggesting that B. glabrata is a suitable test species for freshwater monitoring. Holding procedures and protocols implemented for toxicity and developmental bioassays showed to be in compliance with international standards for intra-laboratory precision. Thereby, we are proposing this system for application in ecotoxicological studies.

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818).

The single cell gel electrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5, 5, 10 and 20 Gy of (60)Co gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens.

Cytotoxic and genotoxic effects of 131 I and 60 Co in follicular thyroid cancer cell (WRO) with and without recombinant human thyroid-stimulating hormone treatment.

Normally, differentiated thyroid cancer (DTC) tends to be biologically indolent, highly curable and has an excellent prognosis. However, the treatment may fail when the cancer has lost radioiodine avidity. The present study was carried out in order to evaluate the cytotoxic and genotoxic effects of 131 I and 60 Co and radioiodine uptake in WRO cells, derived from DTC, harboring the BRAFV600E mutation. WRO cells showed a relatively slow cell cycle of 96.3 h with an unstable karyotype containing various double minutes. The genotoxicity assay (micronucleus test) showed a relative high radioresistance to 131 I (0.07-3.70 MBq/mL), independent of treatment with recombinant human thyroid-stimulating hormone (rhTSH). For the cytotoxicity assay, WRO cells were also relatively resistant to 60 Co (range: 0.2-8.3 Gy), but with a gradual decrease of viability as a function of time for higher doses (20 and 40 Gy, starting from the fifth to sixth day). For internal irradiation with 131 I, WRO cells showed a decline in viability at radioactive concentration higher than 1.85 MBq/mL; this was even more effective at 3.70 MBq/mL, but only when preceded by rhTSH, in coincidence with the highest level of 131 I uptake. These data show promising results, since the loss of the ability of thyroid cells to concentrate radioiodine is considered to be one of the main factors responsible for the failure of 131 I therapy in patients with DTC. The use of tumor-derived cell lines as a model for in vivo tumor requires, however, further investigations and deep evaluation of the corresponding in vivo effects. Environ. Mol. Mutagen. 58:451-461, 2017. © 2017 Wiley Periodicals, Inc.

In vitro cytotoxic and genotoxic evaluation of peptides used in nuclear medicine (DOTATATE and Ubiquicidin29-41) in CHO-K1 cells.

Micronucleus (MN) assay constitutes a valuable surrogate to the chromosome aberration technique for in vitro testing of the genotoxicity of substances. As test substances, two peptidic compounds (DOTATATE and Ubiquicidin29-41) used in nuclear medicine, were tested for in vitro cytotoxicity and genotoxicity in CHO-K1 cells. None of the compounds showed detectable cytotoxicity (0.5-7.3 ng/mL for DOTATATE and 0.3-4.5 ng/mL for UBI29-41), genotoxicity (0.72, 7.2 and 72.0 ng/ml for DOTATATE and 0.45, 4.5 and 45.0 ng/mL for UBI29-41) or cell cycle changes as compared to untreated controls at the concentrations tested. Statistical analysis showed good concordance between two independent analysts. The results corroborate the notion of the safety of the compounds and present improvements of the in vitro MN assay when performed in a pre-clinical trial context that increase the throughput of small-to-medium testing facilities as an alternative to high content screening systems.

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE.

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl2 and NaHCO3 . The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO.

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.

TOTAL CELL NUMBER AND NUMBER OF THE PRIMARY MESENCHYME CELLS IN WHOLE, 1/2 AND 1/4 LARVAE OF CLYPEASTER JAPONICUS.

Total cell number and number of the primary mesenchyme cells of 1/2 and 1/4 larvae were counted at several developmental stages after hatching in comparison with those of a whole larva, using Clypeaster japonicus as material. To obtain partial larvae, blastomeres were isolated at the 2- or 4-cell stage in Ca-free sea water and cultured in natural sea water at around 23°C. Isolated blastomeres cleaved as in situ, namely, as a part of an embryo. Although each partial embryo tended to spread into a plate, it acquired spherical shape prior to hatching of control whole embryo and developed normally in terms of both developmental rate and morphogenesis. Total cell number of a whole larva was about 620 just after hatching and increased almost linearly until i t reached 1850 at the pluteus stage. A half and quarter larvae contained roughly 1/2 and 1/4, respectively, of the number of cells of whole larva through all stages counted. Numbers of the primary mesenchyme cells in the partial larvae, however, tended to be slightly larger than a half or a fourth of that in whole larva. In whole larva, 35, 50, 56 and 58 was counted at the mesenchyme blastula, early gastrula, late gastrula and pluteus stage, respectively.

Dominant lethal effect of 60Co gamma radiation in Biomphalaria glabrata (SAY, 1818).

The dominant lethal effects of gamma radiation of 60Co in the snail Biomphalaria glabrata were studied. Three groups of 13 wild-type snails were irradiated with single doses of 2.5; 10 and 20 Gy. Crossings were carried out at intervals of 7, 17, 23, 30 and 36 days after irradiation. The dominant lethal effect was observed only at the first crossing occurring 7 days after irradiation with 2.5 Gy. With 10 and 20 Gy, the induction of lethal mutations was detected at 7, 17 and 23 days after irradiation; a dose-response effect was observed. The effect was stronger 7 days after irradiation, decreasing in the succeeding crossings up to 30 days. Cell-killing effects on germ cells were detected in the crossings at 23 days and 30 days after irradiation with 20 Gy. After 36 days, frequencies of malformations resumed background levels; crossing rates partially recovered. These results show that gamma radiation affected all the stages of spermatogenesis. Germ cells at later phases were more sensitive to the mutagenic effect of radiation and the cell killing effects were observed on the youngest cells. This response was similar to the highly homogeneous pattern observed in widely different species and allowed us to estimate some parameters of spermatogenesis in B. glabrata.

Antimuscle atrophy effect of nicotine targets muscle satellite cells partly through an α7 nicotinic receptor in a murine hindlimb ischemia model.

We have recently identified that donepezil, an anti-Alzheimer drug, accelerates angiogenesis in a murine hindlimb ischemia (HLI) model. However, the precise mechanisms are yet to be fully elucidated, particularly whether the effects are derived from endothelial cells alone or from other nonvascular cells. Further investigation of the HLI model revealed that nicotine accelerated angiogenesis by activation of vascular endothelial cell growth factor (VEGF) synthesis through nicotinic receptors in myogenic cells, that is, satellite cells, in vivo and upregulated the expression of angiogenic factors, for example, VEGF and fibroblast growth factor 2, in vitro. As a result, nicotine prevented skeletal muscle from ischemia-induced muscle atrophy and upregulated myosin heavy chain expression in vitro. The in vivo anti-atrophy effect of nicotine on muscle was also observed in galantamine, another anti-Alzheimer drug, playing as an allosteric potentiating ligand. Such effects of nicotine were attenuated in α7 nicotinic receptor knockout mice. In contrast, PNU282987, an α7 nicotinic receptor agonist, comparably salvaged skeletal muscle, which was affected by HLI. These results suggest that cholinergic signals also target myogenic cells and have inhibiting roles in muscle loss by ischemia-induced muscle atrophy.

Heart-specific overexpression of choline acetyltransferase gene protects murine heart against ischemia through hypoxia-inducible factor-1α-related defense mechanisms.

BACKGROUND: Murine and human ventricular cardiomyocytes rich in acetylcholine (Ach) receptors are poorly innervated by the vagus, compared with whole ventricular innervation by the adrenergic nerve. However, vagal nerve stimulation produces a favorable outcome even in the murine heart, despite relatively low ventricular cholinergic nerve density. Such a mismatch and missing link suggest the existence of a nonneuronal cholinergic system in ventricular myocardium. METHODS AND RESULTS: To examine the role of the nonneuronal cardiac cholinergic system, we generated choline acetyltransferase (ChAT)-expressing cells and heart-specific ChAT transgenic (ChAT-tg) mice. Compared with cardiomyocytes of wild-type (WT) mice, those of the ChAT-tg mice had high levels of ACh and hypoxia-inducible factor (HIF)-1α protein and augmented glucose uptake. These phenotypes were also reproduced by ChAT-overexpressing cells, which utilized oxygen less. Before myocardial infarction (MI), the WT and ChAT-tg mice showed similar hemodynamics; after MI, however, the ChAT-tg mice had better survival than did the WT mice. In the ChAT-tg hearts, accelerated angiogenesis at the ischemic area, and accentuated glucose utilization prevented post-MI remodeling. The ChAT-tg heart was more resistant to ischemia-reperfusion injury than was the WT heart. CONCLUSIONS: These results suggest that the activated cardiac ACh-HIF-1α cascade improves survival after MI. We conclude that de novo synthesis of ACh in cardiomyocytes is a pivotal mechanism for self-defense against ischemia.

A non-neuronal cardiac cholinergic system plays a protective role in myocardium salvage during ischemic insults.

BACKGROUND: In our previous study, we established the novel concept of a non-neuronal cardiac cholinergic system--cardiomyocytes produce ACh in an autocrine and/or paracrine manner. Subsequently, we determined the biological significance of this system--it played a critical role in modulating mitochondrial oxygen consumption. However, its detailed mechanisms and clinical implications have not been fully investigated. AIM: We investigated if this non-neuronal cardiac cholinergic system was upregulated by a modality other than drugs and if the activation of the system contributes to favorable outcomes. RESULTS: Choline acetyltransferase knockout (ChAT KO) cells with the lowest cellular ACh levels consumed more oxygen and had increased MTT activity and lower cellular ATP levels compared with the control cells. Cardiac ChAT KO cells with diminished connexin 43 expression formed poor cell-cell communication, evidenced by the blunted dye transfer. Similarly, the ChAT inhibitor hemicholinium-3 decreased ATP levels and increased MTT activity in cardiomyocytes. In the presence of a hypoxia mimetic, ChAT KO viability was reduced. Norepinephrine dose-dependently caused cardiac ChAT KO cell death associated with increased ROS production. In in vivo studies, protein expression of ChAT and the choline transporter CHT1 in the hindlimb were enhanced after ischemia-reperfusion compared with the contralateral non-treated limb. This local effect also remotely influenced the heart to upregulate ChAT and CHT1 expression as well as ACh and ATP levels in the heart compared with the baseline levels, and more intact cardiomyocytes were spared by this remote effect as evidenced by reduced infarction size. In contrast, the upregulated parameters were abrogated by hemicholinium-3. CONCLUSION: The non-neuronal cholinergic system plays a protective role in both myocardial cells and the entire heart by conserving ATP levels and inhibiting oxygen consumption. Activation of this non-neuronal cardiac cholinergic system by a physiotherapeutic modality may underlie cardioprotection through the remote effect of hindlimb ischemia-reperfusion.

Evaluation of the cytogenetic effects of (131)I preceded by recombinant human thyrotropin (rhTSH) in peripheral lymphocytes of Wistar rats.

The present study was carried out to investigate the cytogenetic effects of therapeutic exposure to radioiodine preceded by rhTSH in an animal model. Three groups of Wistar rats (n = 6) were used: one group was treated only with (131)I (11.1 MBq/animal); the other two groups received rhTSH (1.2 mug/rat of either Thyrogen or rhTSH-IPEN, respectively) 24 h before administration of radioiodine. The percentage of lymphocytes with chromosome aberrations and the average number of aberrations and of dicentrics per cell were determined on blood samples collected 24 h, 7 and 30 days after administration of (131)I. The data show that the treatment with radioiodine alone or associated with rhTSH resulted in a greater quantity of chromosome alterations in relation to basal values after 24 h, with a gradual decline after 7 and 30 days of treatment. An increase in chromosome alterations was also seen after rhTSH treatment alone. Neither of the treatments, i.e., with (131)I alone or associated with hormone, resulted in an aneugenic effect or influenced the kinetics of cellular proliferation in rat blood lymphocytes. There was no significant difference between the cytogenetic effects of Thyrogen and rhTSH-IPEN treatment. These data suggest that the treatment with radioiodine, associated or not with rhTSH, affects to a limited extent a relatively small number of cells although the occurrence of late stochastic effects could not be discarded.

Genotoxic and cytotoxic effects of 60Co gamma-rays and 90Sr/90Y beta-rays on Chinese hamster ovary cells (CHO-K1).

Among various types of ionizing radiation, the beta emitter radionuclides are involved in many sectors of human activity, such as nuclear medicine, nuclear industries and biomedicine, with a consequently increased risk of accidental, occupational or therapeutic exposure. Despite their recognized importance, there is little information about the effect of beta particles at the cellular level when compared to other types of ionizing radiation. Thus, the objective of the present study was to evaluate the genotoxic and cytotoxic effects of (90)Sr/(90)Y-a pure, highly energetic beta source-on Chinese hamster ovary (CHO) cells and to compare them with data obtained with (60)Co. CHO cells irradiated with different doses of (60)Co (0.34 Gy min(-1)) and (90)Sr/(90)Y (0.23 Gy min(-1)) were processed for analysis of clonogenic death, induction of micronuclei (MN) and interphase death. The survival curves obtained for both types of radiation were fitted by the exponential quadratic model and were found to be similar. Also, the cytogenetic results showed similar frequencies of radio-induced MN between gamma and beta radiations and the MN distribution pattern among cells did not follow the expected Poisson probability pattern. The relative variance values were significantly higher in cells irradiated with (90)Sr/(90)Y than with (60)Co in all exposure doses. The irradiated cells showed more necrotic cells 72 h and 96 h after exposure to beta than to gamma radiation. In general, the (90)Sr/(90)Y beta-radiation was more damaging than (60)Co gamma-rays. The data obtained also demonstrated the need to use several parameters for a better estimate of cellular sensitivity to the action of genotoxic agents, which would be important in terms of radiobiology, oncology and therapeutics.

Induction of micronuclei by 153Sm-EDTMP in peripheral blood lymphocytes of patients with bone metastases.

The purpose of this study was to evaluate the degree of cytological radiation damage to peripheral blood lymphocytes induced by 153Sm-EDTMP applied for palliation of metastatic bone pain. Blood samples from 16 patients (46-82 years old), 10 without previous radiotherapy and 6 with previous radiotherapy, were collected before and one hour after the administration of a mean activity of 41.7+/-5.8 MBq/kg of 153Sm-EDTMP. Then the lymphocytes were cultured for cytokinesis block micronucleus (MN) assay. The number of MNper binucleated cells (BC) in patients without previous radiotherapy before the treatment was of 0.030 (+/- 0.016) and after one hour 0.035 (+/- 0.013), although we could find inter individual differences. The basal MN/BC of the patients with no previous radiotherapy was similar to the controls. The increment in the percentage of BC with MN was similar in patients with and without previous radiotherapy. The observed mean of MN/BC is equivalent to a dose range of 0.05 to 0.10 Gy of 153Sm-EDTMP in vitro. The relatively low frequency of lymphocyte with micronuclei after the exposure to 153Sm-EDTMP supported the contention that radiation damage in lymphocytes of patients with painful bone metastases is minimal.

Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance.

DNA integrity is threatened by the damaging effects of physical and chemical agents that can affect its function. Nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove damages causing DNA double-helix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproducts, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). The modulation of xpa expression may modify the DNA repair rate in the cell genome, providing a valuable contribution to an understanding of the NER process. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15 clone shows good induction of the XPA protein and total complementation of XP12RO cell deficiency. Overexpression of this protein resulted in UV cell survival comparable to normal control human cells. Moreover, low expression of the XPA protein in these cells is sufficient for total complementation in cellular UV sensitivity and DNA repair activity. These data demonstrate that XPA protein concentration is not a limiting factor for DNA repair.

Comparative in vivo and in vitro study of the cytogenetic effects of 153Sm-EDTMP in lymphocytes of patients with bone metastases.

153Sm-EDTMP is a radiopharmaceutical used in nuclear medicine for relief of metastatic bone pain with promising results, but there are few studies about the effects of 153Sm-EDTMP in human cells. This study was conducted for the evaluation of the cytogenetic effects of 153Sm-EDTMP in blood lymphocytes from patients with bone metastases (without previous radio or chemotherapy), using the chromosome aberration technique. The degree of cytological damage found in in vivo blood cells of patients was compared with those found in in vitro in an adjusted dose-response curve. Blood samples were collected before and 1 hr after the administration of 153Sm-EDTMP(about 42.31 MBq/kg). The frequency of structural chromosome aberration per cell observed in 1 hr samples (0.054+/-0.035 CA/cell) was higher than basal ones (0.031+/-0.026 CA/cell), although this difference was not statistically significant (p= 0.101). For in vitro assay, blood samples were exposed to different concentrations of 153Sm-EDTMP, during 1 hr (0.37-1.11 MBq/ml). An increase in the frequency of chromosome aberration per cell as a function of the radioactive concentration was found. The data were adjusted by linear regression model (Y= 3.52+/-2.24 x 10(-2) + 11.15+/-3.46 x 10(-2) X). The frequency of aberration/cell found in vivo was 0.054 and for the same activity in vitro was 0.098, this difference being statistically significant (p = 0.02). This result may be related to blood clearance, osteoblastic activity and individual variability. For a more accurate analysis, the study of more donors is necessary.

Estudos ultraestruturais de embriäo de Biomphalaria glabrata (Say, 1818) / Ultrastructural studies of Biomphalaria glabrata (Say, 1818) embryo

Os estudos ultraestruturais de embriöes de Biomphalaria glabrata (Mollusca: Gastropoda), um importante caramujo vetor da esquistossomose näo tem sido explorados. No presente trabalho foi avaliada a técnica mais adequada para o processamento dos embrioes para a microscopia eletrônica. A técnica que forneceu resultados bastante promissores foi a fixaçäo dupla em glutaraldeído 1% mais tetróxido e ósmio 1% em tampäo cacodilato 0,05 M (pH 7, 4) a 4-C, pré-contraste em acetato de uranila1% durante uma noite e a embebiçäo tanto em resina EPON como Polylite sob vácuo. Foram utilizados embrios no estádio de trocófora jovem que se caracteriza pela intensa organogênese. Alguns aspectos da ultraestrutura de células embrionárias de B. glabrata säo apresentados